1. Field of the Invention
The invention relates to a method for conducting bacterial IMViC tests. More particularly, the invention relates to an improved method suitable for conducting multiple bacterial IMViC tests on solid media.
2. Description of the Prior Art
The abbreviations used herein are listed in the following table:
TABLE 1 ______________________________________ C. diversum or freundii Citrobacter diversum or freundii E. aerogenes or agglomerans Enterobacter aerogenes or agglomerans EC or E. coli Escherichia coli EMB eosin methylene blue IMViC indole, methyl red, Voges-Proskauer, citrate K. ozaenae, pneumoniae or Klebsiella ozaenae, pneumoniae or rhinoscleromatis rhinoscleromatis LST lauryl sulfate tryptose ______________________________________
Many isolation methods and identifying tests have been developed to determine the identity of bacterial strains. The isolation methods are designed to take advantage of the different growth requirements of the various families of bacteria. By using specific culture (growth) media, it is possible to obtain a culture of bacteria which has a limited number of bacterial strains. These strains may be differentiated further by using appropriate tests. These tests are formulated to take advantage of the various metabolic requirements and products of the large number of known bacterial strains. Thus, using a specific isolation method and a series of tests it is possible to identify an unknown bacterial culture as a specific genus, species and variety. Four such known identifying tests are the indole, methyl red, Voges-Proskauer and citrate tests which are collectively known as the IMViC tests.
The indole test is used to detect the production of indole by the bacterial sample. A positive (+) result indicates the presence of indole in the test medium and is determined by the development of a dark red color upon the addition of the appropriate reagent to the test medium. The methyl red test determines the acidity or basicity of the medium after prolonged incubation of the bacterial sample. A red color which develops upon addition of methyl red indicator to the medium is indicative of an acidic medium and represents the positive result. The bacterial product of acetyl-methyl-carbinol is detected using the Voges-Proskauer test. The positive result is the production of acetyl-methylcarbinol and is indicated by a red color which develops upon the addition of specific reagents to the medium. The growth of bacteria with citrate as the only carbon source is the basis of the citrate test. Bacterial growth on the citrate agar is a positive result and is also indicated by the change in color of the medium from greeen to deep blue. If these results do not occur, the tests are said to be negative.
The IMViC tests are the most important and useful tests for differentiation of coliforms, i.e. bacteria of the genera Escherichia and Enterobacter, into species and varieties. Furthermore, the IMViC tests are used generally for distinguishing members of the family Enterobacteriacea and as a basis for the primary differentiation of E. coli and Enterobacter aerogenes. For example, E. coli type 1 yields a ++-- result respectively to the IMViC tests whereas Enterobacter aerogenes yields a --++ result respectively to the IMViC tests. The IMViC tests are also useful for confirming the presence of E. coli in foods. See for example Speck, M. L., Compendium of methods for microbiological examination of foods, American Public Health Association, Washington, D.C. (1970).
However, the IMViC tests have a severe shortcoming which limits their usefulness particularly for confirming the presence of E. coli in foods. This shortcoming is that the IMViC tests are time consuming and procedurally laborious when conducted as prescribed by standard methods. While the indole test requires an incubation of 24 hours and the Voges-Proskauer test requires 48 hours, the methyl red and citrate tests require a minimum incubation of 96 hours. Furthermore, an additional 4 days is required to obtain a suitable culture of the sample bacteria before the IMViC tests may be performed. This length of time makes it impractical to examine the presence of E. coli in foods. The prior art IMViC tests are useful, however, for the testing of coliforms where time is not so critical, e.g. fecal coliform determinations. See for example Speck, M. L. supra and Association of Official Analytical Chemists: Official Methods of Analysis, 11th Ed., Ed. Horwitz, W., Association of Analytical Chemists, Washington, D.C. (1970).
Several methods have been developed which reduce the time of incubation for the methyl red and Voges-Proskauer tests. The Voges-Proskauer test was reduced to only a 6 hour incubation by using a massive inoculum of bacteria in 2.0 ml of test medium and standard test reagents. See for example Coblentz. L. M., Am. J. Public Health 33, 815 (1943). The time of incubation required for both the methyl red and Voges-Proskauer tests has been reduced to less than 18 hours by using small volumes of the test media. See for example Benjaminson, M. A., De Guzman, B. C. and Weil, A. J., J. Bacteriology 87, 234 (1944) and Barry, A. K. Bernshon, K. L., Adams, A. P., and Thrupp, L. D., Applied Microbiol. 20, 866 (1970). It has been proposed to perform the citrate test using a test plate rather than the conventional agar slant. This approach yields a positive test for Enterobacter within 24-48 hours. See Difco Manual, 9th Ed., Difco Laboratories, Detroit, Mich. (1953). However, none of these methods have been widely accepted or followed. Even if these methods are being followed, they do not provide a simplified test method in which all of the IMViC test media are solid and thus capable of being in one test plate as well as reducing the time of incubation as is disclosed in the present invention.
Another limitation of the prior art is the requirement of four separate tubes to complete the IMViC tests using the conventional methods. This means the laborious procedure of separately handling, labeling, inoculating and incubating the requisite number of tubes. Four separate tubes were required by the prior art since a combination of liquid and solid test media were used. While the citrate test utilized a solid agar, the indole, methyl red and Voges-Proskauer tests utilized liquid broths. It has been reported that the indole test could also be performed using a solid agar. See Roche Diagnostics, Improved Enterotube, Roche Diagnostics, division Hoffman-La Roche Inc., Nutley, New Jersey (1974). No method has been proposed which would allow the methyl red and Voges-Proskauer tests to be performed using solid test media. Thus, no method has been proposed which would eliminate the necessity of using separate tubes for these specific individual test procedures.
In order to perform the IMViC tests a suitable culture of bacteria must first be obtained. The isolation procedure generally followed by the prior art is as follows. LST broth was inoculated with a sample of bacteria and incubated at 35.degree. C. for 24 hours allowing the bacteria to grow. Next, EC broth was inoculated with a sample of bacteria from the LST culture and incubated at 45.5.degree. C. for 24 hours. Then Levine EMB agar was inoculated with a bacterial sample from the EC culture and incubated at 35.degree. C. for 24 hours. Difco Manual, supra, which contains a description of the composition of the above broths and agars is incorporated herein by reference. An isolated colony was transferred to a nutrient agar slant and incubated at 35.degree. C. for 24 hours. This last step was required to grow a large enough culture of bacteria to inoculate the four separate tubes required for the IMViC tests as described above. Each tube, using the conventional method, required a separate inoculation thus a minimum of four inoculums were required for the IMViC tests. Therefore, a single colony of bacteria from the EMB agar would not suffice.
U.S. Pat. No. 3,205,151 and Roche Diagnostics, supra, describe an inoculation device and method for inoculating several compartments or tubes of test media utilizing only a single inoculum. This consists of a series of tubes containing the solid test media in an end to end arrangement. An inoculating needle extends through the center of tubes. A single inoculum is picked up with needle and the needle is pulled through all of the tubes inoculating each test media. Since only one colony is required for the inoculation, this colony could be obtained from an EMB culture without the need of growing the bacteria in a nutrient agar or broth. However, these references only disclose the use of a single inoculum, and do not disclose using solid methyl red and Voges-Proskauer test medium.
The IMViC test method of this invention yields at least equally reliable and precise results as obtained with the cumbersome and laborious methods of the prior art. For example, using the improved method of this invention E. coli type 1 yields the same ++-- result as obtained with the prior art. In addition, the improved method is designed to be performed in a single test plate containing compartmentalized, solid test media, thereby eliminating the need for separate tubes of the media. The improved method is also designed to require substantially less time by reducing the incubation time of the methyl red and citrate tests and by reducing the time necessary to obtain a suitable bacterial culture. Thus, at least as equally reliable and precise results are obtained within 48 hours using the improved IMViC test method of this invention when compared to the methods of the prior art. And in fact, the prior art methods need more time to yield the results which are obtained within 48 hours by this invention.